The blood-brain barrier (BBB) acts as a signaling and transport interface between the blood and brain. The BBB begins to form early in embryonic development as the mesoderm-derived vasculature invades the immature central nervous system (CNS) and begins to gain BBB characteristics such as tight junctions and a lack of fenestrae. After further maturation, the adult BBB, with its very low permeability and a wealth of molecular transport systems, is maintained by interactions with supporting cells of the neurovascular unit. While recent studies have indicated the importance of Wnt/beta- catenin, angiotensin II, and sonic hedgehog signaling on BBB development, many BBB attributes remain unaffected when these pathways are disrupted. Thus, our understanding of the molecular mechanisms underpinning BBB formation is incomplete, and in this proposal we aim to further examine the mechanisms that regulate BBB development and maintenance. Recently, we have identified nuclear receptors RXRalpha and PPARdelta as two potential regulators of BBB function. As described in the preliminary data, these receptors are specifically expressed at the BBB compared to peripheral endothelia, receptor agonists can drive BBB phenotypes in endothelial cells differentiated from human pluripotent stem cells (hPSC-derived BMECs), and endothelial-specific deletion of these receptors results in partial neonatal lethality (RXRalpha) and a leaky BBB (PPARdelta) in vivo. Thus, we hypothesize that the nuclear receptors RXRalpha and PPARdelta are crucial regulators of BBB development and maintenance. To test our hypothesis, we will evaluate the in vivo BBB phenotype upon embryonic and postnatal endothelial-specific deletion of RXRalpha and PPARdelta in mice. The molecular mechanisms governed by RXRalpha and PPARdelta activation will be evaluated using the differentiation process of hPSC-derived BMECs as a window to human BBB induction and maintenance. Finally, potential synergy on BBB formation arising from RXRalpha and PPARdelta co-activation will be assessed in vivo and in vitro. Understanding the regulators of BBB induction could yield many new insights regarding fetal brain disease. Furthermore, knowledge of the barrier-genesis and barrier maintenance pathways could open new avenues for restoring BBB function in debilitating neurological disease.